SNPH competes with Trak2 to bind KIF5. (A and B) Representative blots (A) and quantitative analysis (B) showing competition of SNPH and Trak2 for binding KIF5. HEK293 cells were cotransfected as indicated. The KIF5C–Trak2 complex was coimmunoprecipitated (IP) from cell lysates with an anti-Flag antibody and immunoblotted (IB) with an anti-GFP antibody (bottom). Note that SNPH, but not SNPH-ΔKBD, inhibits the formation of the KIF5C–Trak2 complex (red box). Data were quantified from three independent experiments. The asterisk denotes degraded GFP-SNPH. (C and D) SNPH, but not SNPH-ΔKBD, competes with Trak2 to immobilize axonal mitochondria. (E and F) Immunoprecipitation (E) and quantitative analysis (F) showing activity-induced and Ca²⁺-dependent formation of the native SNPH–KHC complex in mature cortical neurons. The cultured neurons (DIV14) were treated for 10 min with 50 mM KCl and 10 µM FPL 64176 in modified Tyrode’s solution with or without 1.2 mM Ca²⁺. The native SNPH–KHC complexes were coimmunoprecipitated from neuronal lysates with an anti-SNPH antibody and subsequently immunoblotted with an anti-KHC and LC8 antibody. Coimmunoprecipitated levels of KIF5 and LC8 after stimulation were normalized against their levels before stimulation, respectively. Note that removing [Ca²⁺]_ext from the medium abolished the activity-induced SNPH–KIF5 interaction. (G and H) Kymographs (G) and quantitative analysis (H) showing enhanced motility of axonal mitochondria after Ca²⁺ depletion. Mitochondrial motility in hippocampal neurons were recorded before and 20 min after removing 1.2 mM [Ca²⁺]_ext from the medium on the same axons (also see Video 9). Bars, 10 µm. Data in D and H were quantified from numbers of axons (indicated in bars). Student’s t test in D and H and Mann–Whitney U test in B and F. Error bars show SEM.